Device for detecting bacterial contamination and method of use

ABSTRACT

A device and method for detecting the presence or absence of a prokaryotic microorganism are provided, comprising the steps of identifying a protein, such as a microbial-specific protease that characterizes the presence of a specific prokaryotic microbe and thereby provides a marker for that microbe; detecting the protease that is a marker for the presence of a specific prokaryotic microbe by cleaving a substrate when the protease is present; and signaling the presence of that protease when cleavage has occurred. More specifically, the method comprises identifying at least one outer membrane protein or a secreted protein that is unique to a particular microbial pathogen such as for example  Listeria monocytogenes  and that is substrate specific.

RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional ApplicationSerial No. 60/201,405, filed May 3, 2000.

FIELD OF THE INVENTION

[0002] The present invention relates to the detection of prokaryoticmicroorganisms. In particular, the present invention relates to a methodand a device for detecting the presence or absence of a prokaryoticmicroorganism or pathogen that contaminants food and food related workareas.

BACKGROUND OF THE INVENTION

[0003] Presently, the United States Department of Food Safety andInspection Services (FSIS) spend over half a billion dollars annually onmeat, poultry, and fish inspections for bacterial contaminants. Broadly,the inspections are used to determine the cleanliness of the work areaand to detect pathogenic microbes in foodstuffs. Ingestion of pathogenicmicrobes can result in food poisoning when the microbes areinadvertently packaged into supermarket goods. One example of apathogenic microbe is Salmonella. Salmonella is a genus of gram-negativebacterium that is a major source of human foodborne illness worldwide.Up to 4 million cases of salmonellosis are reported each year in theUnited States alone. A number of different serotypes of pathogenicSalmonella are known, the most common of which are S. typhimurium and S.enteriditis. Typically, salmonellosis is treated with antibiotics.However, antibiotic resistant strains of Salmonella are emerging.Listeria and E. coli are also commonly occurring microbial contaminants.Listeria monocytogenes is a gram-positive bacterium that is a commoncause of gastroenteritis. Tests for E. coli are performed as anindication of fecal contamination of food and work areas.

[0004] The food industry has tended to test for food contamination atthe production and wholesale level only. However, between the time afoodstuff is packaged and consumption occurs, a bacterial pathogen thatwas undetectable at the wholesale check can multiply and can thus becomea health hazard. Contaminated food is increasing in importance for theproducer. Bad publicity and product recalls cost the food industrymillions of dollars each year.

[0005] Currently, the assay systems used at the wholesale and productionlevel require specialized training and equipment, are time consuming,and are not very sensitive to the presence of small numbers ofpathogenic bacteria. To assay for suspected bacterial contamination, aculture is obtained using a sample of the food or from the workplacethat is suspected of being contaminated in order to increase the amountof detectable substance (suspected bacterial contaminant). Culturing mayrequire up to 48 hours. Then, the cells grown in the culture are lysedto produce a lysate. Historically, assaying for pathogenic bacteria hasbeen done using antibodies in an immunoassay for detecting bacterialproteins in the lysate. In recent years, some companies have used apolymerase chain reaction (PCR) based methods to replicate microbialgenetic material for use in detection assays. The isolated geneticmaterial is multiplied and identified in separate steps. The PCRdetection system also requires hours rather than minutes to perform.Testing is performed at the time of or before shipping a product to thesupermarket. A simple, sensitive, and rapid method for detecting foodcontamination is required both for testing at the wholesale level and atthe retail level.

[0006] Detection devices are also needed at the retail level to protectthe consumer. Testing apparatus useful at the wholesale level generallyis not useful at the retail level for various reasons. First, mostwholesale level tests require technical skills not often practiced bythe typical consumer. Second, when a product remains on the shelf beforepurchase, contaminant levels can increase, especially if the temperatureof the product is not appropriately controlled. During the time the foodis in transit, bacterial growth can occur. Methods and devices fordetecting microorganisms in food at the retail level have been setforth. While tests have been developed for the retail market, they havefailed to be adopted. Colorimetric detectors of changes in ioniccontent, for example pH, of the liquid or material surrounding the foodproduct have been used on or incorporated into retail packaging such asillustrated in U.S. Pat. Nos. 4,746,616 and 5,053,339, the disclosuresof each of which are incorporated herein by reference. Antibodies havealso been coupled to colorimetric labels for detection of contaminatessuch as illustrated in U.S. Pat. Nos. 5,306,466 and 5,869,341, thedisclosures of each of which are incorporate herein by reference. Noneof the aforementioned are sufficiently specific and sensitive to meetthe needs of the current marketplace.

[0007] A means for specifically and sensitively detecting the presenceor absence of at least one pathogenic microorganism in potentiallycontaminated food products at the retail level is needed.

SUMMARY OF THE INVENTION

[0008] The present invention provides a method for detecting thepresence or absence of a prokaryotic microorganism comprising the stepsof identifying a bacterial-specific protein, such as a protease thatcharacterizes the presence of a specific prokaryotic microbe and therebyprovides a marker for that microbe; detecting the protease that is amarker for the presence of a specific prokaryotic microbe by cleaving asubstrate when the protease is present; and signaling the presence ofthat protease when cleavage has occurred. More specifically, the methodcomprises identifying at least one outer membrane protein or a secretedprotein that is unique to a particular microbial pathogen such as forexample Listeria monocytogenes and that is substrate specific.

[0009] Utilizing a bioinfomatic approach, BLAST search comparisons ofthe protease complements of a plurality of common pathogenic bacterialspecies were made. One or more proteases that were unique to a specieswere identified. Next the specific substrate for each protease wasidentified using fluorescence resonance energy transfer (FRET). Theunique, identified substrate was then labeled and used to detect thepresence of a particular microbial pathogen in samples requiringtesting. Identified protease substrates were tested with common foodsand yeast in order to identify substrates that are specific to thebacteria.

[0010] In another aspect, the present invention provides a device fordetecting the marker protein and for notifying a consumer of thepresence of the marker protein. A biosensor for use in retail stores orhome-use to detect contaminated food comprising a specific substratethat is coupled to packaging material proximal to the food stuff to bemonitored for bacterial contamination is provided. Preferably, thesubstrate is covalently bound to a label and thus has a detection signalthat upon proteolysis of the substrate-label bond indicates the presenceof the contaminating bacteria. The biosensor is made by firstdetermining the specific substrate of a specific protease characteristicof the bacteria to be detected. The determined specific substrate islabeled with a plurality of chromatogenic or fluorescent leaving groups.Most preferably, the labeling group provides a latent signal that isactivated only when the signal is proteolytically detached from thepackaging. Chromatogenic leaving groups are, for example,para-nitroanalide groups. Preferably, labeled substrate molecules arecoupled to the interior surface of the packaging material proximal tothe foodstuff. Should the foodstuff become contaminated, the microbialpathogen produces protease which acts on the labeled substrate that isattached to the packaging, liberating the leaving group. When a visuallydetectable colorimetric signal is released, this results in a visiblecolor change at the site of interest.

[0011] In another embodiment, proteases that are common among pathogenicmicrobial species, but that are substrate specific and that share acommon substrate, are identified for use in a method and a device todetect the presence of bacteria. For example, bacteria can collect onwork surfaces in meat plants and in restaurants. A substrate that can becleaved by a plurality of bacteria is labeled and covalently bound to acollector substrate, such as cotton fibers on the tip of a swab. Theswab tip is used to wipe the surface suspected in being contaminated bybacteria. The swab tip is placed in a medium and incubated usingconditions that allow proteolysis of the labeled substrate if theprotease(s) specific for the bound, labeled substrate is present.Proteolysis results in the production of a detectable signal, forexample a color change, may be visible if the signal is a chromatogenicleaving group.

[0012] In yet another embodiment of the present invention, a stripsensor is provided. Preferably, the strip sensor comprises an inertmaterial to which a substrate having a latent detection signal has beencovalently attached. A single protease substrate or a plurality ofprotease substrates may be utilized. When a plurality of proteasesubstrates are utilized, each may be labeled so as to distinguish itfrom another and/or each may be localized in a particular region on thestrip support material. Depending upon the foodstuff, one can predictthe frequency of expected occurrence of a particular pathogenic microbe.For example, Listeria monocytogenes is a common contaminant of poultryproducts, therefore, a chromagenic leaving group labeled substratespecific for a Listeria monocytogenes protease may be incorporated ontothe surface of the packaging proximal to the poultry product. However,where multiple possible microbial pathogens may be present, a substratethat may be proteolyzed by all of the microbes may be utilized as longas it is specific to the pathogenic microbes.

[0013] In still another aspect of the present invention, a kit isprovided for detecting microbial food contaminants. The kit comprises aflat solid support, preferably having a plurality of wells, to which aprotein substrate labeled with leaving groups is linked. A means forproviding a phosphate buffered solution, a negative control, and apositive control are provided. A sample of suspect food (SF) is preparedin phosphate buffer. An aliquot of each of SF, negative control, andpositive control is placed in its own well and allowed to react. Thosewells where a color change is observed contain microbial contaminantunless the negative control well also changes color. If the negativecontrol well changes color, then the assay may be repeated.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 is an image of the Protein Encapsulated Styrofoam (PES) forcapturing bacteria such as L. monocytogenes;

[0015]FIG. 2 is a graph showing the dissolving time of PES tablets asdetermined by the amount of antibody released with time; and

[0016]FIG. 3 is an image that demonstrates the fluorescence activationof a substrate in the presence of L. monocytogenes.

DETAILED DESCRIPTION

[0017] One of the most important considerations for developing awholesale test kit is the ability to capture microbial pathogens infoods. It is necessary to have a procedure for reliably capturingbacteria from food matrices in such a way that all of the viablebacteria are captured and enriched in a suitable recovery medium. Manycompanies use cotton swabs or filters to capture the bacteria, butseveral independent studies have shown that entrapment of bacteria inthese porous materials reduces the accuracy of detecting low-levelcontamination. Standard filter capture methods also entrap bacteria butare less reliable than straight-forward plating, and culture methodstake up to a week to obtain results. Bacterial capture devices such asmagnetic beads are more costly and require an additional step to collectthe bacteria along the wall of the tube with a magnet.

[0018] In food safety testing, the food sample is ground up in a specialblender called a stomacher blender, which is fitted with a sterilestomacher bag. The basic principle of the inventive capture method is toadd a PES tablet to the blended sample for a few minutes with gentleshaking to capture the bacteria. The shell remains intact during thisshort exposure time at room temperature. After the bacteria arecaptured, the PES is allowed to float to the top of the stomacher bagfrom which it is removed with sterile forceps. The tablet is placed ingrowth enrichment medium and incubated at 37° C. until sufficientbacteria can be reliably detected. After sufficient time at 37° C., thegelatin coat dissolves and the bacteria are effectively released intothe growth culture medium.

[0019] To form the PES tablet, gelatin was heated to 65° C. and allowedto cool to about 55° C. Then 50 μg/ml of antibody was added just beforethe gelatin capsule formed around the pellet. The PES technology can beused to capture and release bacteria from a test sample into enrichmentmedium. The rigidness of the gelatin prevents the antibodies from beingreleased until several hours after incubation (see FIG. 2).

[0020] Two PES tablets were incubated in PBS with vigorous shaking atroom temperature (RT; series 1 and 2). At each time point (0, 5,10, 20,60,120, and 720 minutes), an aliquot of the sample was removed and theamount of HRP conjugated antibody was detected with TMB-peroxidasesubstrate (KPL, Gaithersburg, MD). The samples were read with aBenchmark microplate reader at 655 nm. After 20 minutes, less than 1% ofthe total antibody on the PES tablet has dissolved into the PBSsolution. Thus it is possible to capture bacteria with theantibody-coated capsules and release them into the enrichment media withthis method. The rigidness (high bloom number) of the pig skin gelatin(300 bloom) makes this capture and release procedure possible. Using alower bloom gelatin would allow for the bacteria to be released fasterinto the enrichment media.

[0021] The present invention provides a method to quantitatively captureand release contaminating bacteria present in a food sample thatutilizes the protein-encapsulated Styrofoam (PES) tablets describedabove. One or more tablets coated with commercially available antibodiesentrapped in gelatin can be placed in a homogenized food test sample ina stomacher bag and incubated with gentle shaking at room temperature.The tablet can then be removed with sterile forceps and placed in growthmedium at 37° C. for a short period of time, until the gelatin coatdissolved and released the bacteria into the medium.

[0022] The presence of L. monocytogenes contamination is detected bymeasuring the metalloprotease (mpl), which is found only in thepathogenic species of L. monocytogenes (Domann et al., Infection andImmunity. 1991. Jan;59(l):65-72; Mengaud et al., 1991). A series ofpeptides, Ml (lot 038-13/15) and P1 (lot 038-10/12), for use assynthetic substrates for mpl (Table 1) was designed and the sequencesmanufactured by New England Peptide (195 Kimball St., Fitchburg, MA).The peptides were synthesized with fluorescent groups (DABCYL and EDANS)in such a way that the active sites were not blocked. The M1 peptide isspecific for L. monocytogenes at pH 5.5. TABLE 1 First set of peptidesused as probes for FRET analysis of mpl activity Peptide Amino acidsequence MW Description M1 DABSYL-NMLSEVERE-EDANS 1642 Specific toListeria at pH 5.5 P1 DABSYL-ACCDEYLQTKE-EDANS 1838 Substrate with broadpathogen specificity P2 DABSYL-ADTVEPTGAKE-EDANS 1653 Not cleaved byListeria or E. coli

[0023] Preliminary evidence indicates that at pH 5.5, the M1 peptide isspecific for L. monocytogenes and does not significantly detect thepresence of E. coli (see FIG. 3). It is expected that this peptide andderivatives of this peptide (with one or two amino acid substitutions)could be used for a standard microplate assay format for L.monocytogenes in foods.

[0024] It was discovered that Listeria monocytogenes secretes at leasttwo characteristic proteases, one of which is a metalloprotease and onethat is a serine protease. Microbial proteases can be separated intofour major classes based on the catalytic site (metallo, serine,cysteine, and aspartate proteases). The particular type of catalyticsite may be determined using catalytic site blockers. For example thepresence of a metalloprotease having a metallo catalytic site can bedemonstrated by specifically blocking the catalyzed reaction with TAPI[N-{D,L-[2-(hydroxaminocarbonyl)m-ethyl]-4-methylpentanoyl}L-3-(2inapthyl)alanyl-L-alanine,2-aminoethyl amide]. The catalytic site of each protease has acharacteristic amino acid signature, motif, or amino acid sequence thatcan be used to identify that protease and similar new proteases within aclass thereof. In addition, many of the proteases have two forms: alarger inactive form that requires partial proteolysis (limitedproteolysis) to obtain a smaller active form of the enzyme. Proteasesare known to break the peptide bonds in a protein substrate. Someproteases are known to be very specific with regard to the type ofpeptide bond that they are able to hydrolyze or break. When a proteaseis limited with respect to the bond(s) it can break, the protease issaid to be substrate specific.

[0025] The amino acid sequence characteristic of a particular enzyme orof its catalytic site is reproduced from the genetic code. Knowing theamino acid sequence of an enzyme or of its catalytic site, one candeduce the nucleotide sequence in the DNA or RNA that codes for thatsequence. The nucleotide sequence can then be located in the geneticcode of a bacterium or it can be synthesized. Cloning of the geneticsequence coding for the amino acid sequence can be used to providemultiple templates for generating copies of the enzyme or its catalyticsite. Using these copies of the enzyme or its catalytic site, the exactsite of cleavage or type of bond hydrolyzed can be determined. Usingthis information, a specific substrate of the enzyme can be defined.This substrate can be used to assay for the presence of the pathogenicbacteria. The substrate is provided with a label that serves to signalthe presence of the pathogenic bacteria. The preferred label ischromagenic and produces a visual signal when cleaved. For example, whendetermining specific cleavage of a substrate, the substrate can beprovided with two different dyes, where one serves to quench the otherwhen the dyes are in close proximity and measured by FRET.

[0026] Fluorescence resonance energy transfer (FRET) is the process of adistance dependent excited state interaction in which the emission ofone fluorescent molecule is coupled to the excitation of another. Atypical acceptor and donor pair for resonance energy transfer consistsof 4-[[4-(dimethylamino)phenyl]azo]benzoic acid (DABCYL) and5-[(2-aminoethylamino]naphthalene sulfonic acid (EDANS). EDANS isexcited by illumination with 336 nm light, and emits a photon withwavelength 490 nm. If a DABCYL moiety is located within 20 angstroms ofthe EDANS, this photon will be efficiently absorbed. DABCYL and EDANSwill be attached to opposite ends of a peptide substrate. If the peptideis intact, FRET will be very efficient. If the peptide has been cleavedby the metalloprotease, the two dyes will no longer be in closeproximity and FRET will be inefficient. The cleavage reaction can befollowed by observing either a decrease in DABCYL fluorescence or anincrease in EDANS fluorescence (loss of quenching).

[0027] Alternative methods for providing a visual signal includelabeling the protease substrate with a food safe dye (FD&C) such asTexas red or with amido black or with a fluorometric dye such as4-nitroanaline or 7-amino-4-methyl coumarin. For retail applications,labeled substrate may be bound to the food packaging. When the proteasecharacterizing the pathogenic microorganism is present, the label or thelabeled substrate is cleaved from the packaging, diffusing from itsattached site. This results in a color change at that site. For example,the packaging may have “DO NOT EAT” printed in red thereon. When thelabeled substrate is present, the writing is masked. When the label isremoved, the warning becomes visible.

[0028] In one embodiment, an assay system that detects and signals thepresence of predetermined prokaryotic microorganisms is provided.Detection of Listeria monocytogenes is exemplified. However, theinvention is not intended to be limited to that pathogenic species. Thepresence of the predetermined prokaryotic organism of interest isindicated by proteolytic cleavage of a substrate by a protease thatresults in the production of a signal. Preferably, the signal ischromogenic. L. monocytogenes (DPL1955) was kindly provided by Dr. JulieTheriot of the Stanford School of Medicine. The L. monocytogenes wasmade avirulent by a deletion of the gene required for intracellularmotility (ActA, Smith et al., J Cell Biol. 1996 Nov;135(3):647-60).

[0029] A method for developing an assay for detecting a pathogenicbacteria that produces at least one extracellular protease and a methodfor using the assay to detect pathogenic bacteria producing at least oneextracellular protease follows:

[0030] Step 1) Define a catalytic marker amino acid sequence thatuniquely identifies the prokaryotic microorganism of interest.

[0031] Select an amino acid sequence, also termed a marker sequence,that uniquely characterizes or marks the presence of the microorganismof interest, such as, for example, an amino acid sequence unique to anextracellular protease produced by a pathogenic prokaryoticmicroorganism of interest. The selection is performed utilizing abioinfomatic approach. BLAST search comparisons among common pathogenicbacterial species with respect to known amino acid sequences are made.One or more amino acid sequences that are unique to a specificprokaryotic microorganism are determined.

[0032] Step 2) Obtain sufficient protease to determine conditionsfacilitating optimal proteolysis by the protease.

[0033] Isolate the protease from the extracellular medium in which thepathogenic bacteria to be assayed is growing.

[0034] Alternatively, if the genetic sequence for the protease or thelocation of the genetic sequence for the protease are unknown, isolateand clone the genetic sequence that results in the expression of themarker amino acid sequence of Step 1, or, first determine the geneticsequence, and then proceed as before.

[0035] Step 3) Determine the conditions for growth of the prokaryoticorganism and for the production of extracellular protease.

[0036] Determine medium required for growth of the specific prokaryoticmicroorganism of interest and for expression of its unique activeprotease into the medium.

[0037] Determine whether a second protease is required to convert thespecific protease from an inactive precursor form to an active form.

[0038] To determine if the protease has been secreted in an active form,a sample of the bacterial culture is provided with chosen potentialsubstrates and cleavage of these substrates is determined. For example,a bacteria pellet from Step 3 is gently resuspended in 500 μl of DMEM inthe presence of common extended substrates (myosin II, bovine serumalbumin, collagen, fibronectin, and hemoglobin). Myosin II, but not theproteins from milk such as casein, has been shown to be a suitablesubstrate to measure the proteolytic activity of gram positive bacteriaisolated from dried cured ham (Rodrigez et al., J Appl Microbiol. 1998.Nov;85(5):905-12). The suspension of bacteria and substrates isincubated at 37° C. with gentle shaking. At preset times (0.1, 0.3, 1.0,3.0, 5.0, 24, and 48 hours) the samples are centrifuged to spin down thebacteria, and a small aliquot is removed for a SDS-PAGE gel sample.After completion of the time course the samples are run on a 10-15%gradient SDS-PAGE minigel. Then, the proteins are transferred toImmobilon Pseq (Transfer buffer, 10% CAPS, 10% methanol pH 11.0, 15 Vfor 30 minutes) using a Bio-Rad semi-dry transblotting apparatus.Following transfer of the proteins, the blot is stained with Coomassieblue R-250 ( 0.25% Coomassie Brilliant Blue R-250, 50% methanol, 10%acetic acid) and destained (high destain for 5 minutes, 50% methanol,10% acetic acid; low destain until complete, 10% methanol, 10% aceticacid) followed by sequencing from the N-terminal. Alternatively, thesamples will be run on a mass spectrometer in order to map the sites ofproteolytic cleavage using a Voyager Elite Mass spectrometer (PerceptiveBiosystems).

[0039] For example, an extracellular metalloprotease is produced by L.monocytogenes, designated mpl. The gene for this protease is justdownstream of another virulence factor, the listerolysin gene (Domann etal., Infection and Immunity. 1991. Jan;59(l):65-72). Mpl has two forms,active and inactive. The three possible mechanisms for activation of mplor any inactive protease by limited proteolysis are: auto-digestion(autocatalytic processing), digestion by a bacterial protease other thanthe metalloprotease, and digestion by an extracellular host cellprotease. Metalloprotease activity has been found to be indirectlymeasurable in the supernatants of L. monocytogenes. Mpl activity can bemonitored by measuring the shedding of IL-6 receptor from the surface ofhuman monocytes. Processing of the precursor metalloprotease of L.monocytogenes to the mature form was determined to require the presenceof the monocytes.

[0040] The following method can be used to test activation of aninactive protease of other pathogenic bacteria. Preincubate the bacteriaof interest with either methanol fixed whole cells or NaOH strippedmembrane preparations of monocytes. Fixation of the monocytes willprevent the bacteria from internalizing. Briefly, 10,000 CFU of L.monocytogenes is added to nearly confluent dishes of monocytes that arefixed with minus 20° C. 100% methanol for 10 minutes or 5.0%paraformaldehyde in a buffer that preserves the cytoskeleton. After 24hours of incubation, the supernatant containing bacterium is removed andis centrifuged to separate the supernatant from the cell pellet. Thesupernatant is incubated with chosen protein substrates for preset timesto determine its activity.

[0041]L. monocytogenes expression of active metalloprotease was found tobe enhanced by growing the prokaryotic bacteria in a protein free mediumsuch as D10 medium, a medium developed by Trivett and Meyer (1971). Thebacteria were cultured overnight (24 hrs); centrifuged at 5K×g for 10minutes and then resuspended in the tissue culture medium (Dulbecco'sModified Eagle Medium, “DMEM”, Life Technologies (Gaithersburg, Md.).

[0042] Step 4) Identify any specific protein and/or peptide substrate(s)of the active species-specific protease.

[0043] Step 5) Increase the specificity of the protease-substrateinteraction (optional) by determining the active or binding site of theprotease, then determining the genetic sequence useful for producingthat active or binding site, and cloning the determined geneticsequence.

[0044] Identify the target site(s) also termed the “active site(s)” forcleavage by the protease.

[0045] Determine the peptide sequence for the active site of theprotease using fluorescence resonance energy transfer (FRET).Fluorescence resonance energy transfer (FRET) is the process of adistance dependent excited state interaction in which the emission ofone fluorescent molecule is coupled to the excitation of another. FRETis one of the best ways to identify the substrate specificity for aparticular protease (Liu et al., Anal Biochem. 1999. Feb15;267(2):331-5). A typical acceptor and donor pair for resonance energytransfer consists of 4-[[4′-(dimethylamino)phenyl]azo]benzoic acid(DABCYL) and 5-[(2iaminoethylamino]-naphthalene sulfonic acid (EDANS).EDANS is excited by illumination with 336 nm light, and emits a photonwith wavelength 490 nm. If a DABCYL moiety is located within 20angstroms of the EDANS, this photon will be efficiently absorbed. DABCYLand EDANS will be attached to opposite ends of a peptide substrate. Ifthe peptide is intact, FRET will be very efficient. If the peptide hasbeen cleaved by the metalloprotease, the two dyes will no longer be inclose proximity and FRET will be inefficient. The cleavage reaction canbe followed by observing either a decrease in DABCYL fluorescence or anincrease in EDANS fluorescence (loss of quenching).

[0046] When the enzyme is a metalloprotease (mpl), the active form ofthe mpl can be amplified by polymerase chain reaction using forward andreverse primers containing useful restriction sites for cloning into anexpression vector with a strong inducible promoter (such as the T7system of pET-28a, Novagen). Useful primers for the amplification arecatgccatgggtagaacgggctgataccca (SEQ ID NO. 4) andgcgccggaattctcagttaaccccaactgctt (SEQ ID NO. 5). Each primer has atleast a 6 base pair overhang. The DNA is amplified from either lysedcolonies of L. monocytogenes or from purified preparations of genomicDNA from L. monocytogenes. The forward primer is provided with a Nco Isite that includes the start methionine (CATATG). The reverse primerincludes a stop codon (TGA) and an Eco RI restriction site (GAATTC). Atypical amplification protocol comprises amplifying the genetic sequencewith Taq polymerase under the following conditions: 92° C. initial melt4 minutes (25 cycles, 50° C. anneal 30 seconds, 72° C. extension 1.5minutes, 92° C. melt 30 seconds), 72° C. final extension 10 minutes and4° C. store. The annealing temperature may be increased or decreasedsomewhat to account for the predicted melting temperature (Tm) of theprimers. Following cloning of the genetic sequence for the activemetalloprotease into pET28a, the gene is sequenced by double strandedprimer walk sequencing on an ABI310 genetic analyzer. The recombinantactive form of the metalloprotease genetic sequence is purified bynickel-nitrilotriacetic acid chromatography by taking advantage of thepolyhistidine tag in the pET-28a vector.

[0047] Step 6) Provide a device with a peptide substrate having alabeled carboxy terminus where cleavage thereof has been determined tobe specific by the FRET assay of Step 5 to the protease of thepathogenic bacteria of interest.

[0048] For example, a chromagenically labeled peptide could be bound toa PVDF membrane with a hydrophobic area. When the peptide is clipped,the dye would concentrate in the hydrophobic area, becoming visible.Alternatively, the peptide could be labeled fluorescently, andvisualized with a UV lamp. Another possibility is a peptide with achromagenic leaving group that upon removal changes color. The peptidemay be labeled with a blue dye, coupled to a red surface. To theobserver, the surface would look black unless the specific bacteria werepresent to provide protease that would cleave the peptide, turning thesurface red as the peptide was proteolysed and diffused away. Althoughconventional leaving groups such as 4-nitroanaline or7-amino-4-methyl-coumarin can be quantified spectrophotometrically andfluormetrically, it may be best to use a leaving group such as Texas Redthat has an intense blue color. By placing two or three Texas Red groupsnear the end of the peptide, it would be possible to detect visually theclearance of the substrate by L. monocytogenes protease activity.

[0049] The cysteines at carboxyl terminus of the peptides were labeledwith Texas Red bromoacetamide using a standard procedure described forlabeling actin. See Wang and Sanders, 1990. An example is a hydrophobicpeptide labeled with 2-3 Texas Red molecules. Another possibility wouldbe to label the peptide with Amido black. As the substrate isproteolysed by mpl, the black peptide diffuses away, and a clear zonewould appear indicating the presence of L. monocytogenes.

[0050] The peptide can be coupled to a surface such as a glassmicrotiter plate or a polypropylene food packaging material in theproper orientation. The microtiter plate should provide a plurality ofderivatized binding sites for coupling to the peptide substrate such assuccimidyl ester labeled primary amine sites on derivatized plates(Xenobind plates, Xenopore).

[0051] By maintaining a neutral pH, the amino terminus of the peptidecan be preferentially labeled. Briefly, the peptide (at a concentrationof 1-3 mg/ml) will be incubated with a Xenobind plates for 2 hours in 50mM sodium phosphate buffer, (pH 7.0). Following rinsing with washbuffer, the plates will be blocked with 3% bovine serum albumin for 2hours at 37° C. Finally the plates will be rinsed three times in washbuffer and stored at 4° C. until use.

[0052] Optionally unoccupied reactive sites on the microtiter plate oron the food packaging are blocked by coupling bovine serum albumin, orthe active domain of p26 thereto. p26 is an alpha-crystallin typeprotein that is used in this case to reduce non specific proteinaggregation. The ability of the p26 protein to refold heat denaturedcitrate synthetase before and after coupling to the surface of the foodpackaging is used as a control for determining p26 activity.Alpha-crystallin type proteins were recombinantly produced usingstandard recombinant DNA technologies (Maniatis et al., Molecularcloning: a laboratory manual. 1982). Briefly, the plasmid containing thebeta sheet-charged core domain of p26 is electroporated intoelectrocompetent B121 (DE3) cells (Bio-Rad E. coli pulser). The cellsare grown up to an OD of 0.8, then induced with 1 mM IPTG for 4 hours.The cells are spun down, and sonicated in low buffer (10 mM Tris, pH8.0, 500 mM NaCl, 50 mM Imidizole) to lyse (Virsonic, Virtis, Gardiner,N.Y.). The lysate is spun down at 13,000×g for 10 minutes, and thesupernatant 0.45 and 0.2 μm filtered. This filtrate is loaded onto aNi-NTA superose column (Qiagen, Valencia, Calif., cat #30410). Highbuffer (10 mM Tris pH 8.0, 500 mM NaCl, 250 mM Imidizole) is used toelute the protein.

EXAMPLE 1 Rapid Listeria Monocytogenes Detection at the Wholesale Level

[0053] Food products were tested in pentuplicate to determine the rangeof applicability for ready to eat food products. Each solid food samplewas processed in a laminar flow hood. Twenty-five grams of each foodproduct were removed from its package and weighed. The weighed sample ofthe solid food was placed in a sterile stomacher bag with 125 ml ofphosphate-buffered saline (PBS, 10 mM potassium phosphate, pH 7.4, 120mM sodium chloride, 2.7 mM potassium chloride). Solid food washomogenized in a stomacher blender (Fisher Scientific; Pittsburgh, Pa.)and the filtered supernatants were used for testing. L. monocytogeneswas captured and efficiently released by adding a plurality of ProteinEncapsulated Styrofoam Tablets. PES tablets (described above) wereincubated with the food extract for at least a 30 minute incubationperiod. The PES tablet were removed from the surface and placed inenrichment media to efficiently release the bacteria. After incubatingat 37° C. for at least 4 hours, the media was spun down at 5,000×g for10 minutes, and the pellet was resuspended in 50 μl of PBS. Liquid foodswere prepared by mixing an aliquot of the liquid food with one of thePES tablets without stomaching the liquid. After the 30 minute capturestep, the PES tablet was recovered and the pellet was placed inenrichment media.

[0054] A protease based assay was performed on each sample. Each well inthe microtiter plate was read before starting the assay in order to getan accurate measurement of the background to control for well to wellvariability. The protease assay was performed using microtiter plateshaving FRET labeled peptide such as Ml that is described above.Typically 0.5 μg of peptide was incubated with 50 μl of bacterialculture extract for at least 20 min at 37° C. In the presence ofbacteria the FRET peptide will fluoresce at 493 nm. As positivecontrols, either L. monocytogenes or the active recombinant form of thepathogen specific protease can be added to some of the food samplesprior to incubation in the microtiter plate assay. Negative controlsconsist of food samples that are determined to be devoid of bacterialpathogens based on existing published methods (Curtis et al., 1995).

EXAMPLE 2 Rapid Listeria Monocytogenes Detection of Bacteria for HomeUse

[0055] PVDF is a blotting membrane that is hydrophobic until soaked inmethanol, when it becomes hydrophilic. A ring of methanol was stamped onPVDF and allowed to air dry. A peptide substrate labeled with an FD&Capproved dye was incubated with the membrane. It only bound to thehydrophilic ring. This membrane was attached to packaging material, forexample the inside of food packages in direct contact with the food. Ifthe peptide is cleaved by bacterial proteases in the food, the dyemolecule will be attracted to the hydrophobic region of the membrane,and concentrate there, indicating that bacteria is present by theformation of color or florescence within the center of the ring.Bacteria other than Listeria monocytogenes can be detected using thedescribed method and device once a specific protease substrateinteraction has been determined and optimized for the bacterium ofinterest.

[0056] For some bacterial species, it may be necessary to provide amethod of optimizing substrate-protease interaction. For example,divalent cations may be required to optimize protease cleavage of thecoupled substrate. In this case, the food packaging surface will beblocked with proteins and/or peptides that can be precharged with metalions, such as for example polyhistidine or zinc finger motifs.

[0057] One of ordinary skill in the art will appreciate further featuresand advantage of the invention based on the above-described embodiments.Accordingly, the invention is not to be limited by what has beenparticularly shown and described, except as indicated by the appendedclaims. All publications and references cited herein are expresslyincorporated herein by reference in their entirely

1 5 1 9 PRT Listeria monocytogenes 1 Asn Met Leu Ser Glu Val Glu Arg Glu1 5 2 11 PRT Listeria monocytogenes 2 Ala Cys Cys Asp Glu Tyr Leu GlnThr Lys Glu 1 5 10 3 11 PRT Listeria monocytogenes 3 Ala Asp Thr Val GluPro Thr Gly Ala Lys Glu 1 5 10 4 30 DNA Listeria monocytogenes 4catgccatgg gtagaacggg ctgataccca 30 5 32 DNA Listeria monocytogenes 5gcgccggaat tctcagttaa ccccaactgc tt 32

What is claimed is:
 1. A method for detecting the presence or absence ofa prokaryotic microorganism in a sample, the method comprising the stepsof: a. identifying a protease that is unique to the prokaryoticmicroorganism; b. providing a quenched labeled substrate specific forsaid protease; and c. providing the sample; and d. determining thepresence or absence of a detectable label.
 2. The method of claim 1wherein the quenched label is selected from the group consisting offluorescent labeled peptide and colorimetric labeled peptide.
 3. Themethod of claim 2 wherein the means for determining is a colorimeter orfluorimeter.
 4. A method for detecting a plurality of pathogenicmicroorganisms in a sample, the method comprising the steps of: a.identifying a protease that is unique to the prokaryotic microorganism;b. providing a quenched labeled broad spectrum substrate for saidprotease; c. providing the sample; and d. determining the presence orabsence of a detectable label.
 5. A method of using broad spectrumfluorescent or colorimetric labeled peptides to recognize a bacterialspecies by detecting the conjugated peptide with a colorimeter orfluorimeter.
 6. A device for capturing and releasing bacteria from solidor liquid extracts comprising protein encapsulated starch or Styrofoam.7. A device for capturing and releasing bacteria from a sample, saiddevice comprising a pellet and a layer of antibodies entrapped ingelatin surrounding said pellet.
 8. A sensor for detection of bacteriain a sample, said device comprising packaging material having a firstside proximal to said sample and having a second side; and a dye labeledsubstrate for the bacteria wherein said dye labeled substrate isattached to said first side.
 9. A method for using an alpha-crystallintype protein comprising the steps of: (a) expressing and purifying therecombinant alpha-crystallin type protein; and (b) adding thealpha-crystallin type protein to a solid phase or a liquid phase assaycontaining a dye labeled peptide in an amount sufficient to reduceproteolysis of said dye labeled peptide.